Lymphohematopoietic engineering using CAS9 base editors

Abstract

Provided herein are methods and systems for targeted gene disruption (knock-out, missense mutation) and targeted gene knock-in in mammalian cells using base editors and guide RNAs (gRNAs) designed to target splice acceptor-splice donor sites. Also provided herein are universally acceptable genetically engineered cells comprising targeted disruptions in immunotherapy-related genes and comprising a CAR/TCR for therapeutic applications.

We claim: 
     
       1. A method for producing a genetically engineered lymphohematopoietic cell, the method comprising
 (a) introducing into a lymphohematopoietic cell:
 (i) a plasmid, mRNA, or protein encoding a base editor fusion protein comprising a deaminase domain fused to a Cas9 nickase domain, wherein the nickase domain comprises a base excision repair inhibitor domain; and 
 (ii) three or more splice acceptor-splice donor (SA-SD) gRNAs having complementarity to a target nucleic acid sequence to be genetically modified, wherein the three or more SA-SD gRNAs each target a separate sequence selected from the group consisting of SEQ ID NOs: 1-15, and wherein sequences targeted include a PDCD1 gene, a TRAC gene, and a B2M gene; and 
 
 (b) culturing the introduced cell under conditions that promote disruption of splice sites targeted by the three or more SA-SD gRNAs, whereby the target nucleic acid sequence is modified by the base editor fusion protein and the three or more splice acceptor-splice donor (SA-SD) gRNAs relative to an untransfected lymphohematopoietic cell, and whereby a genetically engineered lymphohematopoietic cell is produced having reduced expression of TRAC, B2M, and PDCD1 gene products relative to an untransfected T cell. 
 
     
     
       2. The method of  claim 1 , further comprising introducing into the lymphohematopoietic cell one or more gRNAs designed to produce one or more targeted knock-ins or missense mutations, whereby the genetically engineered lymphohematopoietic cell comprises at least one gene knock-out and one or more gene knock-ins or missense mutations. 
     
     
       3. The method of  claim 1 , further comprising introducing into the lymphohematopoietic cell one or more gRNAs designed to produce one or more targeted knock-ins and one or more missense mutations, whereby the genetically engineered lymphohematopoietic cell comprises at least one gene knock-out, at least one gene knock-in, and at least one missense mutation. 
     
     
       4. The method of  claim 1 , wherein the base editor fusion protein is BE3, BE4, or an adenine base editor (ABE). 
     
     
       5. The method of  claim 1 , wherein the lymphohematopoietic cell is a T cell, Natural Killer (NK) cell, B cell, or CD34+ hematopoietic stem progenitor cell (HSPC). 
     
     
       6. The method of  claim 1 , wherein the one or more SA-SD gRNAs are chemically modified to comprise 2′-O-methyl phosphorthioate modifications on at least one 5′ nucleotide and at least one 3′ nucleotide of each gRNA. 
     
     
       7. The method of  claim 1 , wherein the base editor fusion protein and one or more splice acceptor-splice donor (SA-SD) gRNAs exhibit about 50% to about 90% C-to-T conversion efficiency.